CN118684757A - A long-acting calcitonin analog - Google Patents

Abstract

The invention relates to the field of medicine synthesis, and discloses a long-acting calcitonin analogue. The long-acting calcitonin analogue is used for preparing a pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for preventing and treating osteoporosis, deformational bone diseases, painful neurotrophic failure and malignant osteolysis.

Description

A long-acting calcitonin analog

Technical Field

The present invention relates to a long-acting calcitonin analog and application thereof.

Background Art

Calcitonin is one of the hormones that regulates calcium metabolism and inhibits parathyroid hormone. It can significantly reduce bone calcium loss in high-turnover bone diseases, such as osteoporosis, deforming bone disease (Paget's disease), painful neurodystrophy (Sudeck's salmon calcitonin disease) and malignant osteolysis. Its effect on the trunk bones of postmenopausal osteoporosis is more significant than that on the limbs and on high-turnover bone diseases than low-turnover bone diseases. It can inhibit osteoclast activity and stimulate osteoblast formation. Calcitonin can also inhibit osteolysis, thereby reducing pathologically elevated blood calcium concentrations and increasing urinary calcium, phosphorus and blood sodium excretion by reducing renal tubular reabsorption, but serum calcium will not drop below the normal range.

Calcitonin inhibits the secretion of the stomach and pancreas, but does not affect gastrointestinal motility. Clinical trials have shown that this product has an analgesic effect on patients with certain painful bone diseases.

Since calcitonin has a short half-life in vivo, patients need to be subcutaneously administered every day, and patient compliance is poor. The purpose of the present invention is to provide patients with long-acting calcitonin analogs, reduce the frequency of administration, and improve patient compliance.

Summary of the invention

The present invention provides a long-acting calcitonin analog and application thereof.

To achieve the above object, the present invention first provides a compound of structural formula I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the compound, a prodrug based on the compound, or any mixture of the above forms:

Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-AA1(R)-Leu-Gln-Thr-Tyr-Pro-Arg -Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-AA2(1-7 disulfide bond)

Structural formula Ⅰ

Structural formula Ⅰ AA1 is D-type or L-type Lys, or D-type or L-type Dap, or D-type or L-type Dab, or D-type or L-type Orn, or D-type or L-type Dah, or D-type or L-type Dao;

AA2 in the structural formula Ⅰ is NH ₂ , or OH;

In the structural formula I, R is HO ₂ C(CH ₂ ) _n1 CO-(AA3) _n2 -(PEG _n3 (CH ₂ ) _n4 CO) _n5 -, or HO ₂ C(CH ₂ ) _n1 CO-(AA3) _n2 -(AA4) _n6 -, or is absent, wherein:

n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5;

n6 is an integer from 1 to 10;

AA3 is γGlu, or εLys, or β-Ala, or γ-aminobutyric acid, or 5-Ava;

AA4 is Ala, or Gly, or Leu, or Phe, or Ser, or Thr, or Tyr, or Asp, or Glu, or Gln, or Lys, or D-Lys, or Arg, or His.

The long-acting calcitonin analogues of the present invention include pharmaceutically acceptable salts, solvates, chelates or non-covalent complexes, drug precursors based on the compounds, or any mixture of the above forms.

The present invention also provides a pharmaceutical composition comprising the compound according to the present invention, and provides use of the pharmaceutical composition of the compound according to the present invention in preparing a medicament for treating a disease.

Furthermore, the pharmaceutical composition is used for the prevention and treatment of osteoporosis, deforming bone disease, painful neurasthenia and malignant osteolysis.

More contents involved in the present invention are described in detail below, or some of them can also be experienced in the embodiments of the present invention.

Unless otherwise indicated, the quantities of different components and reaction conditions used herein may be interpreted as "roughly" or "approximately" in any case. Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under respective experimental conditions due to different standard errors.

In this article, when there is a disagreement or ambiguity between the chemical formula and the chemical name of a compound, the chemical formula is used to accurately define the compound. The compounds described herein may contain one or more chiral centers, and/or double bonds and structures such as these, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optical enantiomers or diastereomers. Accordingly, any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in its entirety, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereomer) and any mixture of these isomers. These racemic isomers and mixtures of stereoisomers can also be further separated into their constituent enantiomers or stereoisomers by those skilled in the art using continuous separation techniques or chiral molecule synthesis methods.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above cases, single enantiomers or diastereomers, such as optically active isomers, can be obtained by asymmetric synthesis or racemate resolution. The resolution of the racemate can be achieved by different methods, such as conventional recrystallization with a resolving agent, or by chromatographic methods. In addition, the compounds of formula I also include cis and/or trans isomers with double bonds.

The compounds of the present invention include, but are not limited to, compounds of formula I and all of their pharmaceutically acceptable forms. The pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures of the above forms.

DETAILED DESCRIPTION

The present invention discloses a long-acting calcitonin analog and its use. Those skilled in the art can refer to the content of this article and appropriately improve the relevant parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. The Chinese names corresponding to the English abbreviations involved in the present invention are shown in the table below:

English abbreviation Chinese name English abbreviation Chinese name Fmoc 9-Fluorenylmethoxycarbonyl OtBu tert-Butoxy tBu Tert-butyl Boc tert-Butyloxycarbonyl Trt Trityl Pbf (2,3-Dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl Ala Alanine Leu Leucine Arg Arginine Lys Lysine Asn Asparagine Phe Phenylalanine Asp Aspartic acid Pro Proline Cys Cysteine Ser Serine Gln Glutamine Thr Threonine Glu Glutamate Trp Tryptophan Gly Glycine Tyr Tyrosine His Histidine Val Valine Ile Isoleucine Dap 2,3-Diaminopropionic acid 5-Ava 5-Aminovaleric acid Dab 2,4-Diaminobutyric acid Aib GABA Orn Ornithine Ada 2-Aminoadipic acid Dah 2,7-Diaminoheptanoic acid Apm 2-Aminopimelate Dao 2,8-Diaminooctanoic acid Asu 2-aminosuberic acid

Example 1 Preparation of Compounds

The preparation method is a peptide solid phase synthesis method, including: preparing a peptide resin by a solid phase peptide synthesis method, the peptide resin is then subjected to acid hydrolysis to obtain a crude product, and finally the crude product is purified to obtain a pure product; wherein the step of preparing the peptide resin by the solid phase peptide synthesis method is to sequentially connect the corresponding protected amino acids in the following sequence to the carrier resin by a solid phase coupling synthesis method to prepare the peptide resin:

In the above preparation method, the amount of the Fmoc-protected amino acid is 1.2 to 6 times the total molar number of the resin fed, preferably 2.5 to 3.5 times.

In the above preparation method, the substitution value of the carrier resin is 0.3-1.5 mmol/g resin, and the preferred substitution value is 0.6-1.0 mmol/g resin.

As a preferred embodiment of the present invention, the solid phase coupling synthesis method is: the protected amino acid-resin obtained in the previous step is deprotected from the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time of the Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

The coupling reaction requires the addition of a condensation reagent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

The coupling reaction requires the addition of an activation reagent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of the activation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

As a preferred embodiment of the present invention, the Fmoc-removing reagent is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, wherein the mixed solution contains 10-30% (V) piperidine. The amount of the Fmoc-removing reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and the side chain protecting groups and then subjected to oxidative cyclization to obtain a crude product:

Further preferably, the acid hydrolysis agent used in the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA is 80-95%, EDT is 1-10%, and the balance is water.

More preferably, the volume ratio of the mixed solvent is: TFA is 89-91%, EDT is 4-6%, and the balance is water. Optimally, the volume ratio of the mixed solvent is: TFA is 90%, EDT is 5%, and the balance is water.

The amount of the acid hydrolysis agent is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.

The acidolysis agent is used for 1 to 6 hours at room temperature, preferably 3 to 4 hours. The oxidative cyclization is performed using an oxidant such as iodine, _H2O2 or _DMSO , preferably iodine. The oxidant is added in a titration manner and the addition is stopped when the oxidation end point is reached.

Furthermore, the crude product was purified by high performance liquid chromatography and freeze-dried to obtain a pure product.

1. Synthesis of peptide resin

Take the carrier resin, remove the Fmoc protection and couple the reaction, and sequentially connect the protected amino acids corresponding to the sequence to obtain the peptide resin:

(1) Insertion of the first protected amino acid in the main chain

Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, slowly add it to the protected amino acid DMF solution under stirring, and stir the reaction at room temperature for 30 minutes to obtain an activated protected amino acid solution for use.

Take 0.01 mol of Rink amide MBHA resin (substitution value is about 0.4 mmol/g), use 20% PIP/DMF solution to deprotect for 25 minutes, wash and filter to obtain the Fmoc-free resin.

The activated first protected amino acid solution is added to the Fmoc-free resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing one protected amino acid is filtered and washed to obtain.

(2) Adding other protected amino acids to the main chain

The same method as the first protected amino acid in the main chain is used to sequentially insert the other corresponding protected amino acids in the main chain to obtain a resin containing main chain amino acids.

(3) Insertion of the first protected amino acid in the side chain

Take 0.03 mol of the first protected amino acid of the side chain and 0.03 mol of HOBt, dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, slowly add it to the protected amino acid DMF solution under stirring, and react with stirring at room temperature for 30 minutes to obtain an activated protected amino acid solution.

Take 2.5 mmol of tetrakistriphenylphosphine palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, deprotect for 4 hours, filter and wash, and obtain the de-Allocated resin for use.

The activated first side chain protected amino acid solution is added to the de-Allocated resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing the first side chain protected amino acid is filtered and washed to obtain a resin.

(4) Insertion of other protected amino acids into the side chain

The same method as described above for inserting the first protected amino acid in the main chain was used to sequentially insert the corresponding protected amino acid and the mono-protected fatty acid in the side chain to obtain a peptide resin.

2. Preparation of crude product

Take the above peptide resin, add a cleavage reagent with a volume ratio of TFA: water: EDT = 95:5:5 (cleavage reagent 10 mL/g resin), stir evenly, and react at room temperature for 3 hours. Filter the reaction mixture using a sand core funnel, collect the filtrate, wash the resin with a small amount of TFA 3 times, combine the filtrate and concentrate under reduced pressure, add anhydrous ether to precipitate, wash the precipitate with anhydrous ether 3 times, and dry it to obtain an off-white powder.

The obtained off-white powder was dissolved in a 20% acetic acid aqueous solution, and a saturated iodine/ethanol solution was added dropwise with stirring until complete cyclization, and the mixture was concentrated under reduced pressure at 35-40°C to obtain a concentrated solution of the crude product.

3. Preparation of pure product

Take the above crude concentrate, filter it with a 0.45 μm mixed microporous filter membrane, and purify it for later use;

The purification was carried out by high performance liquid chromatography, the chromatographic filler for purification was 10 μm reverse phase C18, the mobile phase system was 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of the 30 mm*250 mm chromatographic column was 20 mL/min, the gradient system was used for elution, and the cyclic injection purification was carried out. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, and the main peak was collected and the acetonitrile was evaporated to obtain the purified intermediate concentrate;

The purified intermediate concentrate was filtered through a 0.45 μm filter membrane for later use, and the salt was exchanged by high performance liquid chromatography. The mobile phase system was 1% acetic acid/water solution-acetonitrile, the chromatographic filler for purification was 10 μm reverse phase C18, and the flow rate of the 30 mm*250 mm chromatographic column was 20 mL/min (the corresponding flow rate can be adjusted according to the different specifications of the chromatographic column); gradient elution and cyclic loading method were adopted, the sample was loaded on the chromatographic column, the mobile phase elution was started, the spectrum was collected, the change of absorbance was observed, the main peak of salt exchange was collected and the purity was detected by analytical liquid phase, the main peak solutions of salt exchange were combined, concentrated under reduced pressure, and the pure acetic acid aqueous solution was obtained, and the pure product was obtained by freeze drying.

The following compounds were synthesized using the above method:

Compound 1 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₁ CH ₂ CO-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 2 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₂ CH ₂ CO-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 3 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₃ CH ₂ CO-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 4 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₄ CH ₂ CO-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 5 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₅ CH ₂ CO-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 6 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₆ CH ₂ CO-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 7 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Ser-Gly-Ser-γGlu-20 alkanedioic acid )-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 8 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu- 20Alkanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 9 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser- Gly-γGlu-20 alkanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 10 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-γGlu-20 alkanedioic acid)-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 11 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Gly-γGlu-20 alkanedioic acid)-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 12 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Gly-Gly-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 13 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Gly-Gly-Gly-γGlu-20 alkanedioic acid )-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 14 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(Gly-Gly-Gly-Gly-Gly-γGlu-20 alkane Diacid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 15 Glu-Leu-His-Lys(Gly-Gly-Gly-Gly-Gly-Gly-γGlu-20 alkanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser- Gly-Thr-Pro-NH ₂ Compound 16 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(AEEA-AEEA-γGlu-20 alkanedioic acid)-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Compound 17 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(AEEA-AEEA-AEEA-γGlu-20 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Compound 18 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₅ CH ₂ CO-γGlu-18 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Compound 19 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(AEEA-AEEA-γGlu-18 alkanedioic acid)-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Compound 20 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(PEG ₅ CH ₂ CO-γGlu-22 alkanedioic acid)- Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Compound 21 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(AEEA-AEEA-γGlu-22 alkanedioic acid)-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2

Example 2 Activity determination

1. Detection method

After the Gαs-coupled GPCR is activated by a ligand, it can upregulate the intracellular cAMP level. cAMP acts as a second messenger to mediate a series of cellular responses. cAMP Hunter HEK293- CT-R Gs Cell Line overexpresses the Gαs-coupled CT receptor. The binding of calcitonin (analogs) and the overexpressed CTR receptor upregulates the intracellular cAMP level. The cAMP dynamic 2 Kit can be used to detect the amount of intracellular cAMP produced after calcitonin (analogs) activation, thereby evaluating the biological activity of calcitonin (analogs).

HEK293 cell lines stably expressing CT-R were used to stimulate the stably transfected cells with agonists at different concentrations. The time-resolved fluorescence resonance energy signal of the cells after stimulation at each dose was measured using HTRF technology, and the biological activity of the agonists was then calculated.

2. Measurement results

The results of the measurements are shown in the following table:

Example 3 Determination of preliminary pharmacokinetic properties

The experimental animals were cynomolgus monkeys, and the drug was administered subcutaneously at a dose of 0.1 mg/kg. Blood was collected venously before drug administration (0 h) and 1h, 2h, 3h, 4h, 8h, 12h, 18h, 24h, 48h, 96h, 144h, and 168h after administration. The plasma samples were separated by centrifugation, and the blood drug concentration of the compound in the plasma samples was determined by liquid chromatography-mass spectrometry. The half-life of the compound after subcutaneous (SC) administration is shown in the table below:

Compound t _1/2 (h) Compound 5 53.6

Claims (4)

1. Long-acting calcitonin analogs having structural formula I: Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-AA1(R)-Leu-Gln-Thr-Tyr-Pro-Arg -Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-AA2(1-7 disulfide bond) Structural formula Ⅰ Structural formula Ⅰ AA1 is D-type or L-type Lys, or D-type or L-type Dap, or D-type or L-type Dab, or D-type or L-type Orn, or D-type or L-type Dah, or D-type or L-type Dao; AA2 in the structural formula Ⅰ is NH ₂ , or OH; In the structural formula I, R is HO ₂ C(CH ₂ ) _n1 CO-(AA3) _n2 -(PEG _n3 (CH ₂ ) _n4 CO) _n5 -, or HO ₂ C(CH ₂ ) _n1 CO-(AA3) _n2 -(AA4) _n6 -, or is absent, wherein: n1 is an integer from 10 to 20; n2 is an integer from 1 to 5; n3 is an integer from 1 to 30; n4 is an integer from 1 to 5; n5 is an integer from 1 to 5; n6 is an integer from 1 to 10; AA3 is γGlu, or εLys, or β-Ala, or γ-aminobutyric acid, or 5-Ava; AA4 is Ala, or Gly, or Leu, or Phe, or Ser, or Thr, or Tyr, or Asp, or Glu, or Gln, or Lys, or D-Lys, or Arg, or His. 2. The long-acting calcitonin analog according to claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex, a prodrug based on the compound, or any mixture of the above forms. 3. The long-acting calcitonin analogue according to claim 1 and claim 2, for use in preparing a pharmaceutical composition for treating a disease. 4. The pharmaceutical composition according to claim 3, for use in the prevention and treatment of osteoporosis, osteodystrophy, dystrophic pain and malignant osteolysis.